Widespread allele-specific topological domains in the human genome are not confined to imprinted gene clusters

Background There is widespread interest in the three-dimensional chromatin conformation of the genome and its impact on gene expression. However, these studies frequently do not consider parent-of-origin differences, such as genomic imprinting, which result in monoallelic expression. In addition, genome-wide allele-specific chromatin conformation associations have not been extensively explored. There are few accessible bioinformatic workflows for investigating allelic conformation differences and these require pre-phased haplotypes which are not widely available. Results We developed a bioinformatic pipeline, “HiCFlow,” that performs haplotype assembly and visualization of parental chromatin architecture. We benchmarked the pipeline using prototype haplotype phased Hi-C data from GM12878 cells at three disease-associated imprinted gene clusters. Using Region Capture Hi-C and Hi-C data from human cell lines (1-7HB2, IMR-90, and H1-hESCs), we can robustly identify the known stable allele-specific interactions at the IGF2-H19 locus. Other imprinted loci (DLK1 and SNRPN) are more variable and there is no “canonical imprinted 3D structure,” but we could detect allele-specific differences in A/B compartmentalization. Genome-wide, when topologically associating domains (TADs) are unbiasedly ranked according to their allele-specific contact frequencies, a set of allele-specific TADs could be defined. These occur in genomic regions of high sequence variation. In addition to imprinted genes, allele-specific TADs are also enriched for allele-specific expressed genes. We find loci that have not previously been identified as allele-specific expressed genes such as the bitter taste receptors (TAS2Rs). Conclusions This study highlights the widespread differences in chromatin conformation between heterozygous loci and provides a new framework for understanding allele-specific expressed genes. Supplementary Information The online version contains supplementary material available at 10.1186/s13059-023-02876-2.


Background
Higher-order chromatin conformation forms a scaffold upon which epigenetic mechanisms converge to regulate gene expression [1,2]. Many genes are expressed in an allele-specific manner in the human genome, and this phenomenon is an important contributor to heritable differences in phenotypic traits and can be cause of congenital and acquired diseases including cancer [3,4]. In most cases, allele-specific expression is driven by sequence variants located within gene regulatory elements to confer allelespecific preference for transcription factor binding. Genome-wide association studies (GWAS) have linked variants and diseases and have enabled insights into complex-trait genetics and important biological processes in gene regulation and mechanisms underlying disease. Allele-specific differences in chromatin conformation may be masked by chromatin capture approaches designed to provide a snapshot of a high number of dynamic interactions, averaged across both alleles in a heterogeneous cell population. Such approaches may therefore bias the interpretation of chromatin conformation at sites of allele-specific gene expression (ASE) [5]. Genome-wide association studies (GWAS) have linked variants and diseases and have enabled insights into complex-trait genetics and important biological processes in gene regulation and mechanisms underlying disease [6]. Methods that integrate GWAS data with expression quantitative trait loci (eQTL) data to identify associated genes [7], and approaches that combine epigenetic data such as DNA methylation [8], ChIP-seq, and DNase I hypersensitivity have been used to suggest functional hypotheses for variants associated with diseases [9]. More recently, chromatin interaction information has been used to link GWAS variants to target genes [10][11][12] and more tools are being developed to predict the functional effects of variants in disease including combining artificial intelligence and deep learning with Hi-C data [13].
Genomic imprinting is a special case of allele-specific expression, characterized by parent-of-origin monoallelic expression that is regulated by an array of epigenetic mechanisms rather than genetic sequence of the allele [14]. Epigenetic elements of imprinted gene regulation include sequences that are methylated on only one of the parental alleles (known as differentially methylated regions, DMRs). DMRs further have underlying allelic differences in post-translational histone modifications and "CCCTCbinding factor" (CTCF) occupancy. Where the DMRs have been shown to regulate imprinted gene expression in cis, they are referred to as imprinting control regions (ICRs) (reviewed [15]. Disturbances of the allelic dosage due to chromosomal rearrangements or the epigenetic disruption of co-regulated expression in imprinted genes, lead to defined clinical syndromes collectively known as imprinting disorders (reviewed [15]). The most common imprinting disorders include Beckwith-Wiedemann syndrome (BWS) [16,17] with an incidence of 1 in 15,000 live births, Angelman syndrome (AS, 1:20,000); Prader-Willi syndrome (PWS, 1:25,000) [18], Silver-Russell syndrome (SRS, 1:100,000) [17], Temple (MatUPD14), and Kagami-Ogata (PatUPD14) syndromes [19,20].
The IGF2-KCNQ1 locus, implicated in BWS and SRS, divides into two imprinted gene clusters, each regulated by a separate imprinting control region (ICR). The first is IGF2-H19, with its ICR (H19-DMR) methylated on the paternal allele. The second cluster has a maternally methylated ICR (KvDMR) at the promoter of the long non-coding RNA (lncRNA) KCNQ1OT1 gene [21] that when active silences KCNQ1 and adjacent genes [22]. The SNRPN locus is implicated in Prader-Willi and Angelman syndromes. Imprinting at this region is regulated by a ~35-kb bipartite imprinting control region (PWS-IC and AS-IC). The PWS-IC section is methylated on the maternal allele and is the promoter for the pre-mRNA transcript for SNRPN, SNURF, and SNHG14, which is also a host transcript for several other long and short non-coding RNAs [23]. The DLK1-DIO3 locus is implicated in Temple and Kagami-Ogata syndromes and includes DLK1 and RTL1 (paternally expressed genes) and several maternally expressed ncRNAs (MEG3, RTL1-AS, MEG8), snoRNAs, and miRNAs [24]. Two DMRs, IG-DMR (located in the intergenic region between DLK1 and MEG3) and MEG3-DMR (at the MEG3 promoter), regulate imprinted expression at this locus [25]. These DMRs are methylated on the paternal allele in most somatic tissues.
Imprinted genes are an excellent model system for analyzing epigenetic regulation of gene expression and the study of genomic imprinting has uncovered many paradigms that are generally relevant to gene expression [26]. One such paradigm is that the CTCF can act as a boundary element separating different regulatory elements that could be shared between genes. We and others have shown that differential binding of CTCF-cohesin complexes at the imprinted IGF2-H19 locus regulates access to a series of enhancers through allele-specific differences in higher-order looping interactions [27][28][29][30][31][32].
These early studies used a chromosome conformation capture (3C) technique in which fixed chromatin is digested with a restriction enzyme followed by a ligation reaction that favors regions in close proximity. The principle of 3C technology is that interactions between distant regulatory regions that come close together in the 3D space will be more frequently detected than random interactions [33]. Newer technologies coupled to next-generation sequencing (Hi-C, Capture Hi-C) have enabled the detection of topologically associating domains (TADs), defined as local regions within a chromosome with a high density of interactions (contact clusters) that also exhibit insulation from one another [2,34,35].
TADs are thought to regulate gene expression by increasing the frequency of intradomain promoter-enhancer interactions and insulating against spurious inter-domain interactions. TADs are formed via cohesin-mediated loop extrusion, whereby DNA is bidirectionally extruded through the ring-shaped cohesin complex until it is halted by convergently oriented CTCF to form a TAD boundary [36].
It is further assumed that CTCF and associated protein TAD boundaries compartmentalize the genome to implicitly prevent transcription read-through and spurious transcriptional activation of silent genes or constrain the spread of silencing chromatin [37][38][39][40][41][42]. Parameters such as CTCF density and orientation, as well as DNA methylation, have been shown to affect TAD direction, size and overall structure. Hi-C techniques have also identified that the higher-order structure is further shaped by nucleosome accessibility and divides into A-and B-compartments, each with distinctive chromatin and transcription features.
Mouse models in which an imprinted locus can be deleted and transmitted through either the male or female germline, have enabled allele-specific Hi-C profiles for the Igf2-H19, Dlk1-Dio3 imprinted loci. For these loci in the mouse, it has been shown that the maternally and paternally imprinted genes are located together in large TADs that are similar in both parental alleles. Within the TADs, differential binding of CTCF creates allele-specific subTAD structures that provide the instructive or permissive context for imprinted gene activation during development [43].
A limitation to studying imprinted genes in humans has been the need for family studies to ascertain the parental origin of genes. Technologies that detect long-range cis interactions fortuitously link single-nucleotide polymorphism (SNP) variants within a chromosome and provide molecular haplotype information. One of the first studies to use haplotype phasing in Hi-C data from a human lymphoblastoid cell line, GM12878, detected allele-specific long-range interactions between a distal locus, termed HIDAD (Distal Anchor domain) and the promoters of the maternally expressed H19 and the paternally expressed IGF2 [44]. IGF2-H19 has been studied in great depth as the archetypal locus for allele-specific interactions [44,45]. However, the allele-specific-methylation-sensitive-CTCF-binding-for-alternative-looping paradigm as established for IGF2-H19 is not universally true for all imprinted gene clusters.
In this study, we sought to examine how the higher-order chromatin conformation structures differ between the active and silent alleles at loci containing genes that are allele-specifically expressed in humans. To this end, we assembled a HiCFlow pipeline for processing raw Hi-C data for haplotype phasing and construction of allele-specific chromatin conformation profiles. A number of existing pipelines, including HiC-Pro [46], are capable of performing allele-specific Hi-C. However, these require a pre-phased haplotype as input as they cannot perform de novo haplotype assembly from input Hi-C data. Moreover, most do not have functionality to generate and visualize between-sample normalized differences in contact frequency. As such, we opted to assemble a custom pipeline that integrates the required functionality into a single workflow. Following this, we were able to characterize allelic differences at human imprinted gene clusters to establish the epigenetic framework for differential association frequencies.
Our analyses indicate that imprinted gene domains are not uniformly organized within a canonical higher-order structural profile regulated by elements within the ICRs. At the IGF2-H19 locus, the ICR plays a direct role in directing allele-specific CTCF-mediated higher-order chromatin structures consistent with loop extrusion models, whereas at other loci, the ICR may have indirect or no specific effect. Allele-specific compartmentalization was observed in some cell lines at the SNRPN and DLK1 loci. Rather than remaining spatially and temporally separated from their non-imprinted neighbouring genes, imprinted gene clusters share TADs with nonimprinted genes. Indeed, most allele-specific interactions occur within subTADs. Imprinted domain boundaries may be delimited by TAD structures, but some allelespecific associations can occur across TAD boundaries with concomitant effects of allele-specific expression. Allele-specific interactions were not confined to imprinted domains. In an unbiased genome-wide screen, we detected additional allele-specific TADs (ASTADs). The ASTAD distribution varied between cell lines. We found 8-32% of genes with allele-specific expression to be located within ASTADs. Regions of high genetic variability, such as olfactory receptor loci, the bitter taste receptor (TAS2R) gene cluster and the keratin gene (KRT) cluster, were found to be within ASTADs.

A region capture data set with a HiCFlow pipeline provides an effective platform for haplotype phasing of imprinted gene loci
To examine imprinted loci at high resolution, we first generated a Region Capture Hi-C (RC-Hi-C) dataset in a human breast epithelial cell line, 1-7HB2. This diploid cell line has previously been used to examine allele-specific expression and imprinted methylation for several imprinted genes [31,[47][48][49] and been shown to have methylation and expression profiles consistent with the maintenance of normal imprinting in a somatic cell line. Using a tiled probe RC-Hi-C approach, combined with a frequent 4 base-cutter restriction enzyme (MboI), we generated capture regions (totalling 25Mb) at 5 imprinted chromosomal loci, the largest regions included SNRPN, DLK1-DIO3, and IGF2-KCNQ1 (capture region IDs: CR_3, CR_2 and CR_4, respectively, Additional file 1: Table S1). In total, 34,399 probes were used covering approximately 4.1Mb (~16.1%) of the capture regions.
Our RC-Hi-C dataset yielded approximately 40 million valid read pairs with a mean coverage of almost 1700 read pairs per kilobase and was comparable to published high-resolution Hi-C datasets at the same genomic regions (Additional file 2: Fig.  S1a). We assembled a Hi-C analysis pipeline (HiCFlow) to process raw Hi-C data to normalized matrices and for haplotype phasing (Additional file 2: Fig. S1b and Methods). This enabled the construction of allele-specific chromatin conformation profiles (alleles designated "A1" and "A2"). Separating the allelic profiles at imprinted loci and visually presenting them as A1 and A2 matrices showed subtle allelic differences in contact frequency. Therefore, we added a subtraction matrix function to the HiCFlow pipeline for highlighting interaction differences between alleles. To further emphasize regions with consistent directional bias, the subtraction matrices were denoised using a median filter (Additional file 2: Fig. S1c). We used the IGF2-H19 locus to benchmark the allele-specific subtraction parameters and confirmed that the known allele associations could be robustly detected (Additional file 2: Fig. S1c). The subtraction matrix methodology provides a compromise between false positive calls in regions of low interaction density and missing interactions in regions of high interaction frequencies.
The RC-Hi-C library provided an excellent dataset to test and refine the HiCFlow pipeline. We included regions of non-imprinted genes that could be tested as negative controls. These showed similar profiles when separated into A1 and A2 alleles, and only slight indistinct differences in a subtraction matrix (Additional file 2: Fig. S1d).

Evaluation of bias during HiCFlow genotyping and haplotyping
The HiCFlow pipeline utilizes Hi-C data to perform both genotyping and haplotyping. A potential source of bias in this approach is loss of variant calling accuracy at differentially interacting sites, leading to mistaken homozygosity. Although missed heterozygous variants would not influence phasing accuracy, it would reduce the coverage of the haplotype. To assess the impact of this, we performed genotyping and phasing of GM12878 using HiCFlow and compared the results with the experimentally validated haplotype. In total, 4,267,624 and 4,049,512 variants were identified by HiCFlow and the high-confidence dataset respectively. Of the 3,799,226 loci common to both datasets, there was 99.8% agreement in variant identity.
Phasing accuracy was similarly assessed; in total, 2,147,688 and 2,063,320 phased variants were identified by HiCFlow and the high-confidence dataset respectively. Of the 1,942,361 loci with informative phasing information in both datasets, there was 99.9% agreement in phasing. Visual comparison of the subtraction matrices at the IGF2-H19 locus revealed no substantial differences (Additional file 2: Fig. S2). These results support the appropriateness of using Hi-C data to perform both genotyping and haplotyping. However, where available, the user should provide a list of SNPs generated from a non-biased assay to control for the possibility of missing rare but very strong allele-specific interactions. The HiCFlow pipeline includes a function to enable users to provide their own list of SNPs prior to phasing.
Our RC-Hi-C library captured a 1.2-MB section H19-KCNQ1 domain and included the proximal enhancer to the H19 gene [31], both imprinting control regions (H19-DMR and KvDMR, marked by arrows above the CTCF track in Fig. 1a) and CTCF sites upstream of the OSBPL5 promoter. It does not include the HIDAD region described by Rao et al. [44]. In Fig. 1a, the diploid contact matrix for the region shows a series of dense interactions forming several TADs, against a backdrop of cross-TAD interactions, reminiscent of contiguous TAD cliques [51,52]. The IGF2 gene promoters are located at the TAD boundary on both alleles (Fig. 1a). The allele-specific matrices display marked differences between A1 and A2, particularly at the H19-DMR and the KvDMR regions. In the A1-allele, the H19-DMR falls within a TAD, whereas in the A2-allele the DMR subdivides the TAD. By contrast, the ICR at the KCNQ1OT1 promoter region (KvDMR) forms a weak subTAD boundary. The difference in allele-specific enhancer interactions with IGF2 (labelled 1, in Fig. 1a) and H19 (labelled 2 in Fig. 1a) is most clearly seen as a blue signal for the A1 allele and a red signal for the A2 allele in the subtraction matrix. This fits with the known shared enhancer model for IGF2 and H19 promoters being regulated by the CTCF sites in the H19-DMR [27][28][29][30][31][32].
The subtraction matrix further indicates a mosaic pattern throughout the region rather than an enrichment of associations of one allele over another such as a complete single colored TAD. Instead, allele-specific bidirectional stripes from the boundary at the IGF2 gene and KvDMR regions are evident. At short distances, these stripes show a bias towards the A2 allele, whereas at longer range the bias is towards the A1 allele. At the KvDMR, the A2-allele has bidirectional interactions towards the CTCF sites upstream of the KCNQ1 and CTCF site within the KCNQ1 gene, as well as towards the CTCF sites at the KCNQ1DN, CDKN1C, and PHLDA2 genes.
At the SNRPN locus, our RC-Hi-C library captured a 1.6-MB region including NPAP1 to UBE3A. The bipartite ICR (marked with an arrow in Fig. 1b) is flanked by two CTCF sites. Neither of these sites forms a strong TAD boundary as seen by the weak insulation score below the matrix in Fig. 1b. The strongest insulation score in the region is at the UBE3A transcription unit corresponding to a small TAD. Overall, TADs are weak/not clearly defined, which could be explained by the low number of CTCF binding peaks at the locus. When the matrix is split into A1 and A2 profiles according to the phased haplotypes, the A2 allele has a fewer long-range associations. The subtraction matrix shows that the region has directional allelic biases: most A2-allele associations (red) are c DLK1 locus. For each locus, the full (diploid) contact matrix (binned at 10kb resolution) is presented, showing the average of all interactions. Below the diploid contact matrix is the TAD insulation score [50], the CTCF track, and the gene track, with imprinted genes annotated. The positions of DMRs with imprinting control functions are highlighted with arrows above the CTCF track for each matrix. Adjacent to the diploid matrices are the haplotype phased allele-specific matrices (A1 above and A2 below) and a subtraction matrix highlighting the differences between the alleles. Enrichment of A1 relative to A2 (blue), enrichment of A2 relative to A1 (red), the scale bar represents distance-normalized differences between A1 and A2. A SNP density track is included to indicate areas of reduced SNP densities that cannot be haplophased. Allelic differences in these regions cannot be called. Coordinates refer to genome build GRCh37/hg19 towards the left, whereas A1 (blue) are towards the right. We have highlighted these as triangles with dotted outlines (Fig. 1b). The scarcity of CTCF binding leads us to postulate that the TAD-like structures at this locus may be formed through phase condensation of heterochromatic compartments. However, region capture data is not suitable for analysis of compartments by current available methodologies.
The 1-7HB2 RC-Hi-C library captured a region of ~ 2.5MB around the DLK1-DIO3 locus. In the diploid contact matrix, we identified a TAD domain overlapping the imprinted region (Fig. 1c). The DMRs (both IG-DMR/MEG3-DMR, marked by an arrow) are within this TAD and appear to form a subTAD boundary (Fig. 1c). However, at this resolution, it is also likely that the subTAD boundary is formed by a CTCF site upstream of DLK1. In the individual allele matrices, the A1-allele forms a slightly larger subTAD with CTCF bound region upstream of the DLK1, while on the A2-allele, the subTAD is more clearly anchored at the ICR (Fig. 1c). The subtraction matrix shows a V-shape above the DMR. Towards the DIO3 locus, it forms a predominantly red A2-allele stripe whereas upstream of DLK1 it forms a blue A1-allele stripe.
It has been proposed that ICRs such as the above DMRs, mediate their epigenetic functions by directing allele-specific chromatin conformation. However, not all imprinted genes contain CTCF sites at their ICRs [48]. Allele-specific chromatin conformation in the 1-7HB2 cell line for the above imprinted gene clusters indicate that while the IGF2 locus is very clearly shaped by the H19-DMR (ICR) that regulates CTCF site availability, this is not invariably the case at other loci. Indeed, at the SNRPN locus, TAD-like structures are assembled in the absence of CTCF sites, and independent of the ICR.

Do imprinting control regions directly participate in allele-specific interactions?
To further examine whether known ICRs are anchor points for allele-specific chromatin interactions, we conducted allele-specific viewpoint analyses using haplotype phased Hi-C data from the following cell lines GM12878, IMR-90, and H1-hESC, alongside our 1-7HB2 library. We compared these to the relevant subtraction matrices, to which we also added Peakachu loops [53]. Viewpoint analyses enable focused examination of associations with the ICRs in high-resolution data sets, unobscured by the intrinsic density of Hi-C data. Adding the additional cell line data sets enabled us to investigate how expression, methylation, and heterochromatin compartments correlate with allelespecific chromatin conformation. EBV transformed lymphoblastic cell lines such as GM12878 retain DNA methylation profiles consistent with monoallelic expression for several imprinted genes [54]. GM12878 has been haplotype phased previously, as one of the original International HapMap Project cell lines. The remaining cell lines are karyotypically normal diploid and the publicly available Hi-C data have suitable read depth (Additional file 2: Fig. S1a), to suggest that they could be haplotype phased in our HiC-Flow pipeline.
We first examined the IGF2-H19 and the KCNQ1 loci. In GM12878 cells, the maternal origin of the H19 interactions with the downstream HIDAD locus and the reciprocal paternal IGF2-HIDAD interactions have previously been demonstrated [44]. We used this information to set the paternal interactions as A1 (blue) at the H19-DMR and maternal interactions at the IGF2 promoter regions as A2 (red) in the subtraction Imprinting control region (ICR) conformation at the Beckwith-Wiedemann syndrome locus impact differently on chromatin conformation. a Direct influence of the ICR in structuring local allele-specific chromatin conformation at the IGF2-H19 locus, in GM12878, IMR-90 and H1-hESC. Denoised subtraction matrices show that the CTCF regulated H19-DMR (arrow) subdivides the region (10kb resolution, blue areas correspond to A1, paternal allele, red areas to A2, maternal allele). Below the matrices we have the SNP density, and allele-specific loops, generated by Peakachu (red maternal, blue paternal), that corresponds to maternal and paternal expression of H19 and IGF2 respectively. The color of loops matches the underlying value of the subtraction matrix. The lower panels represent the viewpoint interaction traces for each cell line showing interactions between the H19-DMR and loci 400kb in both directions (blue trace A1, paternal, orange A2, maternal). The H19-DMR is methylated on the paternal allele in normal cells. b Indirect influence of the ICR in structuring local allele-specific chromatin conformation at the KCNQ1 locus, in GM12878, IMR-90 and H1-hESC. Subtraction matrices and viewpoint interaction traces as in a above but focused on the KCNQ1 locus and the KvDMR ICR (arrow), normally methylated on the maternal allele. Subtraction matrices and the allele-specific loops below display variable structural effects on chromatin conformation by the KvDMR. The viewpoint interaction traces mostly show weak biallelic interaction traces for the KvDMR associations. The CTCF site highlighted with an * is "region 3, " previously been shown to be important for allele-specific (maternal) expression of KCNQ1 [21] matrices for all three cell lines. This enabled us to also assign the parental origin to the nearby KCNQ1 locus. Figure 2 demonstrates the effects of the ICRs on higher-order structures at the H19 and KCNQ1 loci. We note that the H19-DMR strongly anchors the maternal allele-specific interactions as can be seen in the subtraction matrices (Fig. 2a). This association is independent of the H19 RNA levels which are high in H1-hESCs, and relatively low in GM12878 and IMR-90 (Additional file 2: Fig. S3a). IGF2 transcript levels in IMR-90 and H1-hESC are higher than in GM12878 cells (Additional file 2: Fig. S3a). Interestingly, the three cell lines vary for the CTCF interactions with the H19-DMR, possibly indicating different tissue-specific enhancer associations. Both GM12878 and IMR-90 show this ICR associating with HIDAD region (~0.3Mbp downstream of the ICR), while in H1-hESCs the ICR interacts with regions further downstream (Fig. 2a). At the H19-DMR viewpoints (Fig. 2a, bottom), IMR-90, and GM12878 show peaks of highfrequency A2 (maternal) associations, 50-200kb downstream of this ICR, which correlates with H19-enhancer sites. H19-DMR also forms weaker biallelic associations at sites up to 150kb upstream. H1-hESCs in contrast have a stronger biallelic association peak upstream of the ICR and fewer allele-specific enhancer peaks downstream, which may reflect less stable imprinted expression previously reported in human ESCs [55]. Overall, despite the variable expression, the subtraction matrices show similar "stripe" structures in all three of the cell lines, which would be consistent with an allele-specific loop extrusion between CTCF sites. Thus, the H19-DMR is an anchor point for a scaffold of stable allele-specific associations at the IGF2-H19 that ostensibly only depend on whether this ICR is correctly methylated.
In contrast at the KCNQ1 locus, the ICR (KvDMR) has a weaker and more variable effect on the higher-order structure (Fig. 2b). Viewpoint analyses at this ICR showed that it formed associations about 200kb upstream and downstream, in all the cell lines tested, albeit weaker than that seen for the H19-DMR and not as allele-specific. In IMR-90 cells, there is a maternal-specific peak (A2-allele) approximately −100kb of the KvDMR (Fig. 2b, bottom) that is also seen in 1-7HB2 (Additional file 2: Fig. S3b). For GM12878 and H1-hESC, the associations with the KvDMR viewpoint are biallelic (Fig. 2b, bottom). This ICR is a promoter for KCNQ1OT1 for which we confirmed the expression in these cell lines as well as in 1-7HB2 (Additional file 2: Fig. S3a). Median levels of methylation at KvDMR are about 50% for IMR-90 and GM12878 (Additional file 2: Fig. S3c) with a pattern of methylated and unmethylated alleles consistent with an expected pattern for allele-specific methylation and expression and confirms our previous reports for 1-7HB2 and IMR-90 [48]. For H1-hESC, overall methylation levels were less than 25% (Additional file 2: Fig. S3c), but still separated into a pattern of methylated and unmethylated alleles, which together with the overall levels suggests loss of methylation and biallelic expression of KCNQ1OT1. Thus, the viewpoint analysis for the IMR-90 cells fits with allele-specific regulation of loops by the ICR. However, for the other two cell lines, there is more ambiguity regarding regulation by ICR.
In all three cell lines, the subtraction matrices indicate that associations surrounding KCNQ1 are predominantly on the maternal allele (Fig. 2b). The CTCF signal is weak at the KvDMR in these cell lines (Fig. 2b), and there is controversy in the literature about whether this ICR contains CTCF binding sites [56][57][58][59][60]. A paternal-specific loop connecting the KvDMR and other CTCF sites was only present in IMR-90. In IMR-90, the KvDMR is the anchor point of a small subTAD and bidirectional allele-specific loops with CTCF sites in KCNQ1 (A2, maternal) and CDKN1C (A1, paternal) (Fig. 2b). We note that in these cell lines, several maternal loops were formed between an intragenic CTCF site within the KCNQ1 gene (marked with an asterisk in Fig. 2b) and other sites at the locus. Recently, this CTCF site has been described as "region 3" by Naveh et al. [21], and it was proposed that interactions between this site and surrounding CTCF sites drive transcription of KCNQ1 and CDKN1C on maternal alleles and are required for normal methylation of the KvDMR. SNPs within this CTCF binding site have previously been reported to be associated with a risk for loss of methylation at this ICR [61]. If this model is correct, then the maternal conformation would prevent KCNQ1OT1 expression. KCNQ1OT1 transcription from the paternal allele could potentially displace the intragenic CTCF binding at KCNQ1 on the paternal allele to reciprocally prevent KCNQ1 and CDKN1C transcription. Thus, unlike the H19-DMR, the KvDMR has an indirect effect on chromatin conformation at the locus.
At the SNRPN locus, viewpoint analysis at the bipartite imprinting control region indicates a low frequency of associations within a +400-kb window from the ICR (Additional file 2: Fig. S4). GM12878 showed an allele-specific interaction with the PWS-AS-IC viewpoint at about +400kb that corresponded with a small A1 enriched TAD-like area on the subtraction matrix. However, allele-specific loops between the ICR and other sites were not identified by the Peakachu algorithm, as shown below the subtraction matrix (Additional file 2: Fig. S4, left). We did not detect similar interactions in the viewpoint plots for IMR-90, 1-7HB2 and H1-hESC, despite the subtraction matrices indicating a strong accumulation of allele-specific associations especially in H1-hESC (IMR-90 due to reduced SNP density at this region is uninformative). In the embryonic cells, there is more CTCF binding at this locus, compared to other cells and a more distinct TAD structure. Unexpectedly, despite this strong difference in the subtraction matrices, and the clonal methylation patterns indicating an allelic split between methylation and unmethylated alleles, the overall methylation data for CpG sites at the ICR indicate that H1-hESC is hypermethylated, suggesting that there is a prevalence of methylated alleles (Additional file 2: Fig. S3c). SNRPN RNA (normally paternally expressed) is present in all these cell lines, but at a lower level in IMR-90. The bipartite imprinting center at the SNRPN locus therefore seems to affect allele-specific chromatin conformation at the wider locus. This effect is not reliant on the methylation status of the DMR region within the ICR, at least not in H1-hESC cells. The substantial allelic interaction differences in H1-hESC, despite hypermethylation of the DMR, may reflect the stability of imprinted gene expression of this locus in embryonic stem cells which occurs independently of DNA methylation [55,62].
At the DLK1-DIO3 locus, only IMR-90 and 1-7HB2 cells showed allele-specific associations with the IG-DMR viewpoints (Additional file 2: Fig. S5, right). The subtraction matrices for IMR-90, 1-7HB2 and H1-hESC show an allele-specific stripe of interactions from the IG-DMR/MEG3-DMR with CTCF sites up to DIO3 (and beyond in the case of H1-hESC). There may also be a stripe in the opposite direction; however, this is less consistent between cell lines. The MEG3-DMR contains CTCF sites, which have been reported to be important in maintaining imprinting in somatic tissues [25]. The strength of allele-specific loops (shown below the subtraction matrices) does not correlate with mRNA levels for DLK1, MEG3, MEG8 RTL1, or DIO3 in these cell lines. Indeed, 1-7HB2 and IMR-90 which had the lowest level of expression for these genes showed the strongest allele-specific loops, and more intense differences between A1 and A2 on the subtraction matrix (Additional file 2: Fig. S5). IMR-90 cells which showed the most distinct difference in allelic conformation was also the only cell line with overall methylation levels likely to support allele-specific methylation (Additional file 2: Fig. S3c). However, in this case, the intermediate levels of methylation could not be validated, as clonal analysis showed that the methylation patterns do not separate into allelic differences. This may be as a result of well-known experimental allele-drop out and clonal artifacts of allelic bisulphite sequencing.
The methylation analysis was done bioinformatically using published whole genome bisulfite sequencing (WGBS) data to provide the mean methylation level per CpG within the region (each dot is a CpG in the boxplot in Additional file 2: Fig. S3c), averaged for all for the region (median line in the boxplot in Additional file 2: Fig. S3c). For an imprinted region, we expect the median methylation score to be 50% if the alleles are methylated on one allele only. However, 50% methylation can also be the result of a heterogeneous mix of methylated CpGs across both alleles. Clonal bisulphite analysis (depicted as circle plots in Additional file 2: Fig. S3c) reveals the methylation state of a CpG in single PCR amplicons. At imprinted loci methylation should separate into patterns of methylated and unmethylated amplicons, but without analyzing large numbers of amplicons, the percentage of methylated to unmethylated alleles cannot be determined. Thus, where alleles separate into methylated and unmethylated amplicons on a circle plot, the box plot could indicate that the ratio of methylated to unmethylated alleles is skewed towards hypomethylation (e.g., KvDMR in H1-hESC and IG-DMR in GM12878, Additional file 2: Fig. S3c) or hypermethylation (e.g., PWS-AS-IC in H1-hESC).
The analyses of these four imprinted ICRs indicate that they participate in chromatin conformation to variable degrees in normal cell lines. At the H19 locus, the H19-DMR robustly directs allele-specific chromatin conformation in keeping with a CTCF-mediated methylation-sensitive enhancer competition model. Here it seems that the chromatin conformation is a stable scaffold even in the absence of H19 or IGF2 expression. The IG-DMR seems to direct the chromatin conformation, when normally methylated. At other loci, the ICRs can have indirect effect on chromatin conformation such as the KCNQ1 and SNRPN loci. At the SNRPN locus, where there is low amount of CTCF binding, allele-specific associations are present but do not seem to be driven by the ICR. These results suggest ICRs utilize a variety of mechanisms in addition to CTCF insulation to facilitate allele-specific chromatin conformation at imprinted loci.

Allele-specific compartment differences and effects of imprinting domains on neighbouring loci in normal cells
It is not yet understood how imprinted domains are contained locally and why they do not spread across an entire chromosome. It is expected that chromatin structural elements and compartmentalization confine imprinted genes to TADs or subTADs to prevent allele-specific associations spreading beyond their domains. To examine how far allele-specific associations spread and to detect A/B-compartments, we added the CscoreTool (v1.1) [63] to our HiCFlow pipeline and examined a wider 3-6Mb window around each imprinted cluster.
At the IGF2-H19 locus, allele-specific interactions did not extend beyond the HIDAD region (chr11:1,500,000) in our cell lines (Additional file 2: Fig. S6a). Interestingly, the recently identified associations between KRTAP5-6 and INS are present within this region [64]. At the KCNQ1 locus, we identified looping interactions extending from the KCNQ1 region to NUP98 and RRM1 in an adjacent TAD in H1-hESC. NUP98 and RRM1 are both monoallelic in this cell line, but are not known to be imprinted, which suggests that monoallelic interactions can and do extend beyond a TAD containing imprinted genes (Fig. 3a, Additional file 2: Fig. S6a). The Cscore analysis for this locus indicates that it is located within a 4-Mb active A-compartment on both alleles in all three cell lines shown as a red bar below the allele-specific matrices in Additional file 2: Fig. S6a.
The SNRPN locus is within a heterochromatin B-compartment that starts upstream of the imprinted MKRN3 locus and extends 5Mb towards the telomeric end of chromosome 15 in the three cell lines (Additional file 2: Fig. S6b and Fig. 3b, top). In GM12878, this is a bifold B-compartment that splits into two sections at a point of insulation just after the UBE3A gene (Additional file 2: Fig. S6b). Subtle allelic differences are noted in the matrices for the A1 and A2 alleles. In A1, just above the PWS-IC, there is a break within the B-compartment which is not present in the A2-allele, which remains within the B-compartment (Additional file 2: Fig. S6b). A similar bifold pattern is seen for this region in IMR-90 cells, except for a region just above the ATP10A locus which shows this gene to be in an A-compartment on both alleles in allele-specific matrices (Additional file 2: Fig. S6b). The A2 allele shows further small interruptions in the B-compartment just above the SNRPN cluster of genes. The A1 allele does not show these breaks (Additional file 2: Fig. S6b). The most striking difference in allele structure for this locus is seen in the embryonic cells (H1-hESC), where the Cscore analyses returns a similar B-compartment for the full matrix as for the other cells, but in the separate alleles the region above the imprinted genes is distinctly located within a wider active A-compartment in one allele (A1), whereas on the A2-allele the imprinted locus remains in an inactive B-compartment (Fig. 3b, top). Interestingly, on the A1 allele, the active compartment seems to spread slightly beyond the boundary upstream of MKRN3. H1-hESC seems to have more distinct TAD structures in the separate allele matrices compared to the other cell lines (Fig. 3b, top, and Additional file 2: Fig. S6b). The subtraction matrices and Peakachu loop algorithm confirm the presence of only a few allele-specific loops in GM12878 and IMR-90 (Additional file 2: Fig. S6b), whereas H1-hESCs have several allele-specific loops, forming a TAD above the SNRPN region (Fig. 3b). There are also several cross-TAD associations between the SNRPN TAD and the adjacent MKRN3 TAD. These results suggest that the SNRPN locus is shaped by phase condensation in conditions of low CTCF binding [65], and that when present, CTCF can enhance and stabilize compartmentalization.
The DLK1-DIO3 locus, which showed the clearest allele-specific differences surrounding the ICR in IMR-90 (Additional file 2: Fig. S5), was found to have allelic differences in Cscores in these cells (Fig. 3b, bottom). The A1-allele of this locus was in a B-compartment whereas the A2 was in an active A-compartment. In GM12878 Fig. 3 Effects of imprinting domains on neighbouring loci and allele-specific compartment differences in normal cells. a An example of cross-TAD associations from an imprinted gene region. The subtraction matrix at the H19-KCNQ1 locus with allele-specific loops in H1-hESC demonstrating cross-TAD association between KCNQ1 region to NUP98 and RRM1 which are allele-specifically expressed (ASE), but not known to be imprinted. Gene density is shown in blue below the CTCF track, with imprinted genes below, and genes with ASE below. b Examples of allele-specific compartmentalization at SNRPN and DLK1-DIO3 loci. The diploid contact matrix (10kb resolution) with a Cscore below (blue for B-compartment, red A-compartment), followed by TAD insulation score, CTCF track, imprinted genes, and ASE genes. Adjacent to the diploid matrices are the haplotype phased allele-specific matrices (A1 and A2). Note the allele-specific differences in the Cscore track between A1 and A2 alleles at both loci. See Additional file 2: Fig. S6 for a comparison of the other cell lines, and subtraction matrices. c Allele-specific cross-TAD associations and additional TAD domains enriched for allele-specific associations near the DLK1-DIO3 locus. Subtraction matrices, SNP densities, allele-specific loops, imprinted and ASE genes are as described. The DLK1-DIO3 domain in H1-hESC and GM12878 forms several cross-TAD associations and has weak TAD boundaries. In H1-hESC several genes adjacent to the imprinted domain have allele-specific expression. In GM12878, a nearby TAD (labelled v-TAD) has stronger enrichment for allele-specific associations than DLK1-DIO3 locus and H1-hESC cells, the locus has no allelic differences in Cscore with both alleles either in a B-compartment (GM12878, Additional file 2: Fig. S7a) or A-compartment (H1-hESC, Additional file 2: Fig. S7a). In IMR-90, the TADs containing the imprinted genes seem to be sharply defined and separate from neighbouring TADs with no overlapping interactions, especially in the B-compartment (Fig. 3b, bottom). In H1-hESC, there seem to be more cross-TAD interactions and less sharp TAD borders, although no loops extend from the imprinted region into other TADs. Several allele-specific expressed genes in this cell line are detected in the A-compartment, including WDR25 and SETD3 located upstream of DLK1 in an adjacent TAD (Additional file 2: Fig. S7a, left). This 1Mb sized TAD contains several ncRNAs as well as coding genes, none of which have yet been reported to be imprinted in human, although there is evidence that one or more of the orthologous genes are tissue-specifically imprinted in mice [66].
All cell lines have a large 3-Mb-sized TAD corresponding to a B-compartment that is located 2Mb upstream of the DLK1 cluster (Fig. 3b, bottom, Additional file 2: Fig.  S7a). The subtraction matrices show strong allelic bias for predominantly A1 associations in GM12878 cells, and to a lesser extent in H1-hESCs (Fig. 3c, right). We have named this the "v-TAD", after a single coding gene, VRK1 near the TAD boundary. We examined this region to see whether structural variations were present, specifically duplications that can skew the ratio of allelic associations and found two duplications of 750 and 89bp (nssv16165643, chr14:98,934,427-98,935,179 and nssv16173610, chr14:97,441,932-97,442,020), that were not associated with any genes and a 304-bp duplication in an intron of the VRK1 gene (nssv16177248, chr14:97,284,401-97,284,704), listed in the NCBI database. In our cell lines, we find no variation in copy number within the v-TAD domain. However, both GM12878 and H1-hESC possess different Indel mutations within the region corresponding to the nssv16165643 duplication. It is unclear to what extent these variants are responsible for allele-specific associations in the v-TAD. Since they do not overlap CTCF sites, we do not anticipate they are responsible for such large-scale allelic changes in GM12878 and H1-hESC.
VRK1 encodes a Serine/Threonine Kinase and is associated with pontocerebellar hypoplasia, Type 1A and Microcephaly-Complex Motor and Sensory Axonal Neuropathy Syndromes. It is widely expressed in several tissues and has roles in cell cycle, mitosis and DNA damage responses. It has never been reported to have monoallelic expression. We found it to be highly expressed in all cell lines (Additional file 2: Fig. S6b) and monoallelic in H1-hESC (Fig. 3c). The v-TAD region in H1-hESC seemed to form several cross-TAD interactions with adjacent TADs, and further genes (PPP4R4 and DICER1) were found to have allele-specific expression (Fig. 3c).
In summary, these results indicate that imprinted regions can have allele-specific associations confined within TADs, without differences in compartmentalization such as at the IGF2-H19 and KCNQ1 loci. We have also seen that compartmentalization can be detected allele-specifically and that imprinted regions when present in active A-compartments can form looping associations that extend beyond their own TAD regions, with the potential of allele-specifically activating genes outside the imprinted locus.

Clusters of allele-specific interactions occur throughout the genome as allele-specific TADs
The detection of the above v-TAD prompted us to examine the frequency in which differences in allele-specific associations can be found within TADs genome-wide. We therefore performed an unbiased ranking of all TADs to assess the allelic association differences genome-wide in GM12878, IMR-90, and H1-hESC cells (Fig. 4a). We defined allele-specific TADs (ASTADs) as having higher than expected absolute differences in A1 and A2 associations. TADs containing imprinted genes (Additional file 3: File S1) had Z-scores of 2.9-5.8 (IGF2-H19, in all three cell lines), 5.5-9.1 The absolute sum of intra-domain allelic differences is calculated. (iv) A Z-score is calculated by comparing against the chromosome-wide background level of absolute differences for a domain of equivalent size. TADs with Z-score > 2 are considered ASTADs. b Venn diagram of conserved ASTADs between cell lines. Conserved ASTADs were defined as any set of domain intervals, between cell lines, that shared 90% reciprocal overlap. c ASTAD enrichment across different chromatin states. Chromatin states are ordered, per cell line, according to their enrichment level as determined by LOLA (max rank). ASTADs possess contrasting enrichment characteristics in H1-hESCs compared the differentiated cell lines. IMR-90 and GM12878 were significantly enriched in active chromatin relative to all TAD domains. H1-hESC were enriched for inactive and bivalent states (DLK1-DIO3 in IMR-90), and the SNRPN locus (2.2-3.1 in H1-hESCs). The v-TAD described above had a Z-score >4 in GM12878 and H1-hESC.
Each cell line had its own unique profile of ASTADs distributed across the genome (Additional file 2: Fig. S8a and b), although there was a small overlap between cell lines (Fig. 4b). Allelic imbalances due to abnormalities resulting in trisomy show up as large blocks of allelic bias, and this was found at the 11q chromosomal region in GM12878 cells (Additional file 2: Fig. S8c). Imbalances due to monosomy will be masked as these regions cannot be phased. Random monoallelic effects such as X-inactivation are not expected to show up as ASTADs because most normal tissues have a 50% mix of cells with either of the parental X-chromosomes inactivated. Indeed, in IMR-90 cells where X-inactivation is random (presumably because it was derived from primary lung tissue rather than cloned from a single cell), no allelic bias was found on the X-chromosome (Additional file 2: Fig. S8d). GM12878 cells, in comparison, have skewed X-inactivation [44], and a large number of allele-specific associations on the X-chromosome (Additional file 2: Fig. S8d). The H1-hESC cell line is male and thus these cells do not register heterozygosity for phasing on the X-chromosome. A full description of all ASTADs detected is available in Additional file 4: File S2. Overall, ASTADs vary in size comparable to non-ASTADs (Additional file 2: Fig. S8e).
To determine whether ASTADs were associated with specific chromatin (heterochromatin compartments, DNAse I sensitivity, histone signatures associated with regulatory elements) or genomic (heterozygous SNPs, lncRNA, CpG islands) features, we carried out locus overlap analysis (LOLA) [67] for 15 states previously imputed using chrom-HMM [68]. This revealed that the ASTADs had different characteristics in H1-hESCs compared the differentiated cell lines. IMR-90 and GM12878 are significantly enriched in active chromatin relative to all TAD domains (Fig. 4c). H1-hESC were enriched for inactive and bivalent states.
CNVs are known to influence chromatin architecture [69] and therefore the detection of ASTADs. CNVs may also influence mappability to the reference genome and introduce artifacts in variant discovery and haplotype phasing. Although HiCFlow implicitly removes CNV-driven biases through HiCcompare, we carried out an independent analysis of CNV using QDNAseq [70] to assess whether ASTADs were associated with non-normal copy numbers. With the exception of a low magnitude, but significant, enrichment of gain CNV in IMR-90, non-normal CNV regions were not substantially over-represented in ASTADs relative to TADs (Additional file 1: Tables S5 and S7).

Conserved ASTADs
The three cell lines tested came from three different individuals and represent three different cellular lineages (lymphoblastic, fetal lung, and embryonic stem cells). Based on these genetic and the tissue differences, we expect only a few ASTADs would be common to all three cell lines. From the above analysis, we found 39 conserved ASTAD. We hypothesized that common ASTADs would fall into two categories. The first category being sequence directed, such that ASTADs present at the same location in the three cell lines would share identical SNPs variants. The second category being ASTADs with stable epigenetic mechanisms directing allele-specific chromatin conformation as in genomic imprinting.
We first tested whether conserved ASTAD boundaries possessed the same, i.e., identical genetic variants, across all three cell lines that may influence chromatin structure in a consistent manner. Randomization testing with repeat random sampling (n = 10,000) of conserved TAD versus ASTAD boundaries (see Methods, Additional file 1: Table S8), indicated significant enrichment (Z-score = 2.13, p = 0.016) of identical genetic variants at conserved ASTAD boundaries compared to conserved TADs.
A similar analysis, examining the distribution of allele-specific methylation (ASM) within conserved TAD versus ASTAD boundaries was performed for each cell line. We did not detect any significant enrichment of in H1-hESC and IMR-90. Enrichment was marginally higher in GM12878 (Z-score = 1,67, p = 0.048). These results suggest that conserved ASTADs are primarily sequence directed due to sharing similar haplotypes.
The conserved ASTAD with the highest ranking allelic difference (Z-score = 4.9-9.7), was identified at chr3:195,270,000-195,730,000 (Additional file 5: File S3). However, this region was found to overlap an ENCODE Blacklist region usually associated with anomalously high read mapping signal [71], possibly due to unannotated repeats in the reference gene sequence. We therefore cannot exclude this ASTAD being an artifact. The next highest ranking conserved ASTADs (chr12:52,530,000-53,390,000, Z-score = 3.6-5.3 and chr7:2,910,000-4,810,000, Z-score = 3.6-5.6) are not within blacklisted regions.
The ASTAD at the chr12:52,530,000-53,390,000 region contains a cluster of keratin type II cytoskeletal orthologues, involved in hair and epithelial keratin synthesis, and a high association with disease-associated variants. This region has been described as an EAFD locus (genetic variants with extreme allele frequency differences) and a feature of such loci are that the SNPs have longer linkage disequilibrium (LD) ranges than random SNPs [72]. KRT1 has been reported to be expressed allele-specifically as a result of cisregulatory polymorphisms [73], and a more in-depth analysis has shown allele-specific expression is a complex trait of multiple SNPs having a cumulative effect on gene expression [74]. In GM12878, IMR-90 and H1-hESCs, none of the KRT genes were listed as having allele-specific expression, despite this region showing strong allelic differences in association frequencies (Additional file 2: Fig. S9).

Imprinted genes in ASTADs
Only 5 Imprinted genes are present in conserved ASTADs. Four of these (H19, IGF2, IGF2-AS and INS) are part of the same imprinted gene cluster on chromosome 11, the other is the recently identified pseudo-gene ATP5F1EP2 [75] on chromosome 13 (Additional file 3: File S1). The IGF2-H19 cluster is the most studied of imprinted genes, and perhaps this is due to its robust and stable CTCF-mediated imprinting mechanism. Little is known about imprinting mechanisms for ATP5F1EP2. However, a close look at this locus showed allele-specific expression of further genes within this ASTAD, including POLR1D, MTIF3, and USP12 in H1-hESC, and RPL21 in GM12878. These genes have not been reported to be imprinted. Gene mutations in POLR1D underlie autosomal dominant inheritance of Treacher Collins Syndrome (TCS, OMIM 154500).
In our targeted analyses, we found that the KCNQ1, SNRPN, and DLK1 loci had several differences between the cell lines and therefore unlikely to be within conserved ASTADs (Additional file 3: File S1). We screened 115 genes reported to be imprinted in humans [76], to determine if they were within ASTADs in any of the cell lines as opposed to being within a conserved ASTAD (Additional file 3: File S1). In H1-hESC, 45 (39%) of the imprinted genes are in ASTADs. IMR-90 and GM12878 have 38 (33%) and 42 (37%) respectively.
Randomization testing (see Methods) revealed that imprinted genes were significantly enriched (p ≤ 0.001) within ASTADs compared to non-ASTADs (Additional file 1: Table S9). However, the observation that so few imprinted genes were in conserved ASTADs suggest that somatic differences in imprinted gene expression, methylation, and other epigenetic effects influence the density of allele-specific interactions such that their TADs do not consistently meet the threshold of a conserved ASTAD.
It has been suggested that polymorphisms within enhancers are more likely to disrupt chromatin architecture and influence gene expression. We therefore assessed whether the heterozygotic variation in enhancers (using public available data from [79]) are enriched in ASTADs and found a significantly higher than expected proportion of heterozygous variants in enhancers overlapping ASTADs (chi-square test, p < 0.001) in GM12878 and IMR-90, but not in H1-hESC. In addition, we find that enhancers associated with ASE genes are significantly over-represented in ASTADs (chi-square test, p < 0.001) in GM12878 (Additional file 6: File S4).
We further found that a cluster of TRIM genes on chromosome 11 contained allelespecific (paternal allele) expressed genes (TRIM5, TRIM22 in GM12878, TRIM6, TRIM6-TRIM34 in IMR-90 and TRIM34, TRIM6-TRIM34 in H1-hESC) and were within an ASTAD in GM12878, and in IMR-90 (Additional file 2: Fig. S6). The ASTAD containing the TRIM cluster also contains a cluster of olfactory receptor genes (OR52 -OR56), which typically express only one allele, but did not feature in the lists of ASE in these cell lines. Olfactory genes have been shown to form interchromosomal associations and aggregate in foci within the nucleus when they are repressed, with the expressed allele localized outside of such foci [80,81].
One ASTAD region of interest included the TAS2R gene cluster that encodes an array of Bitter Taste Receptor genes on chromosome 12p13.2. The TAS2R gene cluster overlaps an ASTAD in both GM12878 (chr12:10,915,000 -11,405,000) and in IMR-90 (chr12:10,900,000-11,370,000) corresponding to an overlap of approximately 90%, but this may be due to a lack of SNP density in IMR-90 (Fig. 5). The ~400kb ASTAD seems to originate from a weak CTCF binding site as a subTAD within a 800-kb-wide CTCF defined TAD. We further found that the region had allele-specific differences in Cscores, such that in H1-hESC, one allele was more enriched for the A-compartment, while the other was divided into several smaller A-and B-compartments (Fig. 5a, right). In GM12878, there was allelic variation within A-compartments, whereas in IMR-90 there was allelic variation within B-compartment. TAS2R genes have not previously been identified as imprinted or to have allelic expression. We examined RNA transcript levels for several of the TAS2Rs genes at the locus by PCR and confirmed that these were expressed in all three cell lines (Fig. 5b). These genes are usually very small single exon genes and despite being in a region of high sequence variability, most of the SNPs are intergenic. Even the lncRNAs, of which there are several at the locus, have very small exons and thus we found no informative SNPs to enable us to verify whether genes at this locus have monoallelic expression. Taste receptors, like olfactory receptors, are G-protein coupled receptors and they may similarly have monoallelic expression due to allelic exclusion. We examined the TASR2 clusters on chromosomes 5 (TAS2R1) and 7 (TAS2R3-TAS2R38) and found that these did not overlap ASTADs. However, to our knowledge this is the first time that taste receptors on chromosome 12 have been reported to be present in an allele-specific chromatin conformation.

Discussion
The recent attempts to link chromatin interaction information with GWAS variants to target genes has led to several tools being developed to predict the functional effects of variants in disease [13]. However, aside from allele-specific differences at imprinted loci, the occurrence of allele-specific TAD structures and their association with allele-specific gene expression has not been extensively documented at a genome-wide level.
In this study, we assembled a novel bioinformatics pipeline, HiCFlow, which combines variant calling and haplotype phasing with allele-specific Hi-C analysis to enable robust investigation and visualization of allele-specific associations. Since genomic imprinting is a phenomenon of epigenetically established parent-of-origin monoallelic gene expression, that is independent of the genetic sequence of the expressed or silenced allele, we initially focused on these genes as domains known to be allele-specifically regulated.
We focused on three imprinted gene clusters that exemplified imprinted domains known to be allele-specifically regulated. The canonical boundary model exemplified by IGF2-H19 is consistent with an allele-specific loop extrusion model, which shows up as a pair of parallel stripes in a subtraction matrix. In this case, CTCF sites anchored at the HIDAD locus (or other sites downstream in a tissue-specific manner) interact with CTCF sites at the H19-DMR on the maternal allele, and with CTCF sites near the IGF2 promoter on the paternal allele.
The key finding at imprinted loci is that although the canonical boundary model for regulating imprinted genes expression as exemplified by IGF2-H19 is consistent with an allele-specific loop extrusion chromatin conformation model, this is not invariably the rule at all imprinted loci. Allele-specific loop extrusion shows up as a pair of parallel stripes in a subtraction matrix at the IGF2-H19 locus and it is the H19-ICR that determines the allele-specific chromatin scaffold. Here, CTCF sites at the HIDAD region and 9 other intervening CTCF pause-sites engage in loop extrusion, either with the CTCFrich H19-DMR on the maternal allele, or with CTCF sites near the IGF2 promoter on the paternal allele. A large body of literature exists for the IGF2-H19 locus, demonstrating that deletion of the ICR, or its inactivation through DNA methylation, results in loss of imprinting, with over expression of IGF2 causing BWS [21,49,[82][83][84][85][86]. It has previously been shown by us and others that allele-specific deletion of CTCF sites in the ICR results in allele-specific conformational changes and that cells from BWS patients with loss of imprinting have allele-specific profiles consistent with "paternalized" conformation structures and that perturbation of methylation profiles using 5-Azacytidine results in restructuring of the chromatin conformation of this locus [21,49,[82][83][84][85]. Thus, the impact of deleting/modifying the ICR on chromatin conformation is known for this locus. In contrast, the adjacent imprinted gene cluster clearly shows that the ICR (KvDMR) regulating imprinting at the KCNQ1 locus has weak if any effects on 3D chromatin structure. If there is allele-specific loop extrusion, this occurs at a CTCF site previously identified as "region 3" and shown to be required for setting up a maternal allele-specific loop to facilitate KCNQ1 expression [21]. The paternally expressed KCN-Q1OT1 lncRNA transcript is regulated by the KvDMR, and its transcription may disrupt CTCF binding at region 3 on the paternal allele to prevent this loop. RNA polymerase 2 has previously been reported to displace CTCF occupancy [87]. However, we have little evidence that this may be the case at the KCNQ1 locus, and even if it did, the ICR would still have an indirect effect on 3D structure at this locus.
Phase condensation models have not previously been tested at imprinted gene loci. Data from chromatin immunoprecipitation for post-translational histone modification profiles at imprinted loci in mouse and humans [88] predict that the silent allele should be in a heterochromatic configuration. Early studies using fluorescent in situ hybridization at the SNRPN locus demonstrated that active and silent alleles occupy different nuclear compartments [89]. Adding the Cscore to the HiCFlow pipeline enabled us to confirm allele-specific differences in compartments at imprinted loci. These differences were undetectable at the IGF2-H19 and the KCNQ1 loci in the cell lines tested. Strong differences in compartmentalization were observed at the DLK1 locus in IMR-90 cells. This correlated with ICR methylation levels that would be consistent with normal imprinting and strong allele-specific association differences. A striking allelic difference in compartment structure was detected at the SNRPN locus in H1-hESC which also showed clear evidence of CTCF occupancy at the locus. Current models suggest that CTCF stabilizes TAD structures and that compartmentalization processes counteract the formation of TADs [90]. Our observations at the SNRPN locus suggest that CTCF stabilized higher-order structures may also be initiated by phase condensation-mediated compartmentalization. Indeed, recent analysis suggests that CTCF may have an instructive function in the formation of condensates [90]. Within imprinted domains, there are also non-imprinted genes that escape the effects of neighbouring gene silencing and we do not yet understand how imprinting is contained locally and why it does not spread across an entire chromosome. By examining the loci from a wider perspective, we found that each locus occurred within a larger TAD structure, which is similar to observations made by the Feil group in mice for the Igf2 and Meg2 loci [43]. The loci we examined suggest that imprinted genes are present within interacting subTADs with weak boundaries. We showed evidence that allele-specific looping associations in imprinting domains that are present in A-compartments can extend into neighbouring TADs. Such observations are rare, and it is feasible that this "neighbor effect" requires that the nonimprinted neighbor is already in an active state and able to be allele-specifically upregulated by associations with enhancers within the imprinted domain.
Our novel HiCFlow pipeline is suitable for processing multi-sample Hi-C, RC-Hi-C and Micro-C datasets. It is implemented as a user-friendly Snakemake pipeline and, to our knowledge, is the first workflow to combine haplotype phasing with allele-specific-Hi-C analysis. Testing it on a RC-Hi-C dataset provides proof-of-concept data for a diagnostic assay that can be used to detect allele-specific chromatin conformation in humans with imprinting disease. In this capture data set, we only examined a small set of imprinted gene clusters, but these could be expanded to the full set of imprinted genes and would still provide the required read depth when sequenced in-house on standard sequencing platforms. A limitation of our RC-Hi-C analysis was that we focused on the core regions of the imprinted domains, which enriched for the detection of shorterrange interactions. We would recommend utilizing tools, such as CHiCANE and Peaky, to detect long-range interactions [91,92]. For the purposes of this study, our focused analysis was sufficient to show that different chromatin conformation structures are present at imprinted loci, rather than a "standard ICR-centered" structure. In the last decade, it has been shown that a subset of patients diagnosed with an imprinting disorder, have multi-locus imprinting disturbances (MLID), characterized by loss of methylation at multiple imprinted loci across the genome (reviewed [15]). RC-Hi-C and HiCFlow will be useful tools in the comprehensive and integrative analysis of MLID.
To investigate the properties of genome-wide allele-specific interactions, we scored each TAD based on the absolute allelic differences observed. The set of top-ranked TADs, denoted "allele-specific TADs (ASTADs), " were assessed for over-representation of various genomic annotations relative to non-ASTADs. Most strikingly, ASTADs were found to be enriched with polymorphic variants. This is unsurprising since heterozygous SNPs are necessary to distinguish alleles during allelic assignment of read pairs. However, enrichment of INDELs in ASTADs, which were not used for allelic assignment, suggests that high genetic variability plays a role in influencing allele-specific chromatin conformation. Indeed, regions of high variability are prone to allele-specific gene expression as demonstrated by the large body of GWAS studies that correlate genetic variants with allele-specific binding of transcription factors, DNA methylation patterns and gene expression (reviewed [93]). This is further supported by our finding that heterozygous variants are more likely to be associated with ASTADs if they overlap a known enhancer.
We also found that allele-specific expressed genes were significantly over-represented in ASTADs in GM12878 (32%) and IMR-90 (18%), although not in H1-hESC (8%). Despite enrichment, only a small absolute proportion of ASE genes overlapped ASTADs. In some cases, it is likely that short-range allele-specific interactions may be indistinguishable at the 20kb resolution of our data. In addition, the absence of informative SNPs can prevent ASE detection and detection of allele-specific chromatin interactions.
As expected, imprinted genes were significantly over-represented in ASTADs compared to non-ASTADs. However, only the H19/IGF2 locus was consistently identified as an ASTAD in all cell lines. Allele-specific associations at imprinted loci also did not correlate with levels of detectable transcripts. It has been suggested that the chromatin conformation forms a scaffold upon which transcription factors can dock and activate gene expression [94]. Such a scaffold would therefore not correlate with expression levels if the right transcription factors are not present. However, while this may be true for some loci, given the overall conserved CTCF binding across multiple tissues and cell types, there is enough variation in TAD structures, and reported experimental evidence indicating that active transcription modulates higher-order chromatin structures [95]. A lack of correlation between looping associations and transcripts could therefore be due to the post transcriptional effects that affect RNA stability. The strength of chromatin loops between an enhancer and promoter and transcription factor binding kinetics has recently been correlated with transcriptional bursting [96,97]. Thus, the frequency and length of time that a gene promoter and enhancer interact affects the frequency and length of a transcriptional burst. Technologies in which such models can be tested experimentally on a genome-wide scale are not yet available.
We were able to confirm that regions of high variability and known to have allelic imbalances in expression such as the olfactory receptor genes were within ASTADs. The TAS2R family of receptors similar to olfactory receptors are G-protein coupled receptors. We were intrigued to find that the bitter taste receptor (TAS2R) clusters were present within ASTADs. To our knowledge, TAS2R genes have not been reported to be subject to allelic exclusion, unlike olfactory receptors. They occur in regions of high genetic variability similar to olfactory receptors. There are about 25 functional TAS2R genes and 11 pseudogenes spread between chromosomes 5, 7, and 11. Extensive population studies have utilized the high variation to examine evolutionary origins of different haplotypes and to identify the selection pressures that an ability to distinguish bitter toxic substances has had on the genetic evolution of this gene family. The functional effects of the variants on G-protein receptor protein structures and the mechanisms whereby they convey taste perception to the brain have been elucidated. However, limited information on their transcriptional regulation exists. It has been recently shown that TAS2R genes are not only expressed on the tongue but that they are more widespread and present in heart and respiratory epithelia as well as in the gut and that they may have further sensing functions unrelated to bitter taste. An in situ hybridization study has indicated that humans co-express a heterogeneous mix of between 4 and 11 taste receptors per cell in papillae of the tongue. Although we do not know if this is due to allele-specific expression, our data indicate that this may be the case as ASTADs are commonly associated with allele-specific expression.

Conclusions
This study exemplifies the utility of the bioinformatics HiCFlow tool for combined variant calling and haplotype phasing with allele-specific Hi-C analysis for investigation of allele-specific associations at regions subjected to epigenetic silencing such as genomic imprinting as well as sequence-mediated influences on expression. Overall, this study highlights how genetic sequence variation and the regulatory mechanisms behind allelespecific gene regulation culminate in widespread allelic differences in chromatin organization that are not confined to imprinted gene loci.

RC-Hi-C library preparation
3-4×10 7 number of 1-7HB2 cells were crosslinked on plate with formaldehyde (Agar Scientific R1026), followed by a quenching step with 1.25M glycine, scraping for cell detachment and two washes with cold PBS 1×. The cell pellet was re-suspended in 50ml freshly prepared ice-cold lysis buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% Igepal CA-630 (Sigma-Aldrich, I8896-50ML), one protease inhibitor cocktail tablet (Roche complete, EDTA-free 11873580001)). Cells were lysed on ice for a total of 30min, with 2 × 10 strokes of a Dounce homogenizer with a 5-min break between Douncing to minimize cell clumping. Following lysis, the nuclei were pelleted and washed with cold 1.25xNEB Buffer 2 (NEB, B7002S) then re-suspended in 1.25xNEB Buffer 2 to make two aliquots of 10-15×10 6 cells for digestion. Hi-C libraries were digested using 1500U MBOI (NEB, R0147M) at 37°C overnight while orbital shaking. Following digestion, the restriction fragment overhangs are filled in for 1h with dNTPs including biotin-14-dATP (Life Technologies, 19524-016). Fragments are then blunt-end ligated with 1U/μl T4 DNA ligase (Invitrogen, 15224-025) under dilute conditions to favor ligation between crosslinked fragments (in a 15-ml tube for overnight at 16°C). DNA crosslinks were then reversed with 10mg/ml proteinase K (Roche, 03115879001) for 6-8 h at 65°C followed by RNase A (Roche, 10109142001) treatment at 37°C for 60 min. Two rounds of DNA extraction/purification were carried out with phenol pH 8.0 (Sigma-Aldrich, P4557) and phenol: chloroform: isoamyl alcohol (Sigma-Aldrich, P2069) followed by precipitation with 3M sodium acetate pH 5.2 (Sigma-Aldrich, S7899) and 2.5× volume of icecold 100% ethanol on wet ice for 1-2 h. The Hi-C Library's quantity and quality were assessed by running 50-100ng of the Hi-C libraries on a 0.8% agarose gel. Hi-C marking and Hi-C ligation efficiency was verified by PCR digest assay using MBOI and CLAI (NEB, R0197S) enzymes. Biotin is then removed from the ends of un-ligated fragments using the exonuclease properties of T4 DNA polymerase (NEB, M0203L), and DNA was sheared to obtain DNA fragments with a peak concentration around 400 bp. DNA ends were repaired and fragments, with internally incorporated biotin, are pulled down using magnetic Dynabeads MyOne Streptavidin T1 beads (Life Technologies 65601). After PE adaptor ligation ((5′-P-GAT CGG AAG AGC GGT TCA GCA GGA ATG CCG AG-3′ and 5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC*T-3), pre-Capture amplification was performed with eight cycles of PCR on multiple parallel reactions from Hi-C libraries immobilized on Streptavidin beads, which were pooled post PCR and SPRI Ampure XP beads (Beckman Coulter, A63881) purified. The final Hi-C library was re-suspended in 25μl of Tris low-EDTA and quantified by the Qubit ™ dsDNA BR Assay Kit (Thermo Fisher, Q32853). The size distribution of the library was assessed by Tapestation D1000 (Agilent). The Hi-C capture regions were enriched via hybridization with biotin-RNA probes (Agilent Technologies). The capture regions of interest were then pulled down with Dynabeads MyOne Streptavidin T1 beads (Life Technologies 65601) and purified using SPRI Ampure XP beads (Beckman Coulter, A63881). Finally, Hi-C Capture library was amplified and then sequenced with Illumina 50 bp paired-end sequencing.

Capture biotinylated RNA oligos design
Capture biotinylated 120-mer RNA oligos (25-65% GC, <3 unknown (N) bases) were designed to target either one or both sides of MBOI site and within 4-500bp as close as possible to the ends of the targeted restriction fragments using a custom genomewide Perl script made available from the Babraham Institute and then submitted to the Agilent eArray software (Agilent) for manufacture.

RNA extraction and qPCR
RNA was extracted from cell lines using TRI Reagent (Sigma-Aldrich) and 1μg of total RNA was converted to cDNA using QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR (qPCR) was performed using a ¼ dilution of cDNA with SYBR Green PCR Master Mix (Thermo Fisher Scientific) on ABI Step One Plus (Applied Biosystems) and specific primers (Sigma-Aldrich) for target genes (see Additional file 1: Table S6).

Explanation of subtraction matrices
Visual comparison of allelic matrices (A1 vs. A2) was performed using "subtraction matrices. " Joint-normalization of raw A1 and A2 matrices was first performed using HiCcompare (v1.6.0) [137]. This provides implicit correction of between-sample bias. Normalized matrices were then transformed using the "Observed / Expected" method to correct for genomic distance and more effectively resolve changes in long-range interactions. Normalized counts were subtracted (A2-A1), and the resulting subtraction matrices were denoised using a median filter (Scikit-Learn v1.7.3) [138]. This emphasizes regions with consistent directional bias and which are more likely to represent signals of interest (Additional file 2: Fig. S1c).

Bioinformatic processing of RC-Hi-C
Processing of RC-Hi-C was as described above, but analysis was restricted to read pairs mapping within a single capture region. All other read pairs were filtered from the analysis.

Compartment analysis
HiCFlow performs compartment analysis using CscoreTool (v1.1) [63]. Compartment analysis was performed on the full Hi-C datasets for each cell line at a 20kb resolution. The sign of the Cscore was oriented such that positive and negative scores represented "A-" and "B-" compartments respectively. Results were intersected with chromatin state data such that negative scores were associated with heterochromatin and positive scores were associated with active transcription ("activeTSS").

Allele-specific TAD (ASTAD) classification
TAD domain detection was performed, using OnTAD (v1.2) [141], on the full Hi-C dataset binned at 10kb resolution. The set of TADs were then used as a reference set to identify TADs with substantial differences in contact frequency between allele-specific matrices. A1 and A2 matrices were first jointly normalized using the LOESS method described in HiCcompare. A median filter was then applied to remove spurious or noisy background interactions. Following this, the absolute sum of differences is calculated within the relevant TAD domain. For each TAD, a Z-score is calculated by comparing against the chromosome-wide background level of absolute differences for a domain of equivalent size. The methodology of ASTAD detection is illustrated in Fig. 4a.

Enrichment analysis
To determine if ASTADs were enriched for particular genomic features, we performed enrichment analysis using LOLA (v1.22) [67]. ASTAD enrichment was tested against a background set of all identified TAD domains in a given cell line. To facilitate cell line comparison, only autosomal regions were tested for enrichment. A full list of genomic features tested and enrichment status is available in Additional file 1: Table S5.

Overlap analysis
Overlap analysis was performed to identify ASTADs that were conserved between cell lines. Conserved ASTADs were defined as sets of ASTAD intervals, between cell lines, with at least a 90% reciprocal overlap. A 10% difference in overlap allows for slight error in domain positioning due the loss of resolution during matrix binning. Given a bin size of 20kb, a 10% difference equates to a shift of approximately one bin length for a median size domain interval. Interval overlap was calculated using BedTools (v2.29.2).

Randomization testing
In each of the following randomization tests, any TAD domain overlapping a region with non-normal copy number or overlapping an Encode Blacklist region were removed from the enrichment analysis.

Comparison of identical heterozygous variants in conserved ASTAD boundaries compared to conserved TAD boundaries
Conserved identical heterozygous variants were defined as any heterozygotic variant (SNPs or Indels) present in all three cell lines. The total number of observed conserved variants overlapping the set of conserved ASTAD boundaries was compared against a null distribution of repeat random samples (n = 10,000) of conserved TAD boundaries. A TAD boundary was defined as the outermost 20kb of a TAD domain, equivalent to 1 bin size on the AS-Hi-C matrices. This analysis was repeated for the allele-specific methylation (ASM) data for each cell line (see Additional file 1: Table S8).

Enrichment of imprinted (or ASE) genes in ASTADs relative to all TAD domains
The total number of observed imprinted genes overlapping ASTADs was compared against an expected distribution of randomly sampled genes. Genes were selected via randomly stratified sampling to match the sample size and distribution of imprinting gene types. Stratification was used due to the imbalance in gene type distributions between the imprinted / ASE gene sets and the total gene sets. Genes were excluded from the analysis if they did not overlap a TAD domain or if they overlapped a blacklisted region or a region with non-normal copy number. A total of 10,000 samples were taken per analysis to build a null distribution and to calculate a Z-score from the observed overlap. This analysis was repeated for each cell line and for ASE genes (see Additional file 1: Table S9).

Methylation status at CpG islands
To check methylation status within regions of interest, preprocessed whole genome bisulfite sequencing (WGBS) data, corresponding to CpG methylation in ENCODE bed bedMethyl format, were obtained from publicly available datasets (GM12878 [142], IMR-90 [143], H1-hESC [144]) as described in "Availability of data and materials" section. Filtering was performed using the methylKit R package [145]. For each cell line, all CpGs overlapping a CpG island were selected. For each target CpG island, boxplot was generated for comparison among three cell lines using the ANOVA statistics.